Hepatitis B Virus (HBV) is endemic worldwide: 2 billion people have been infected with HBV and approximately 400 million people suffer from chronic HBV (1). This year, about 600,000 people will die of HBV-related hepatocellular carcinoma, cirrhosis, and liver failure. The United States alone is home to about 2 million individuals with chronic HBV.
HBV is an enveloped virus with an icoshedral core. The core is assembled in the cytoplasm from core (capsid) protein, viral pregenomic RNA, viral reverse transcriptase, and several host proteins (C. Bourne, et al., 2008, J. Virol., 82:10262-10270). HBV has four open reading frames in its 3200-base pair genome one of which encodes the core protein (HBcAg or Cp), a 183-residue protein 240 copies of which self-assemble as homodimers into a roughly spherical capsid (Bourne, et al., 2008). When packaged with the viral genome and appropriate accessory proteins, the entire complex is referred to as the viral core which plays indispensable roles in viral DNA synthesis (from the pregenome) and intracellular trafficking (Ganem, D., and R. J. Schneider, 2001, Hepadnaviridae: the viruses and their replication, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.). An alternative start codon for the core gene results in expression of the e-antigen, which is identical to core protein except for having an N-terminal extension, a C-terminal truncation, and being secreted into serum as a dimer.
Chronic HBV may vary in its symptoms and its need for treatment. The predominant antiviral treatment strategy against HBV has thus far been to attack viral reverse transcriptase. Not surprisingly, HBV reverse transcriptase inhibitors lead to drug-resistant mutants in HBV (Deres, K., and H. Rubsamen-Waigmann, 1999, Infection 27(Suppl. 2):S45-S51; Tennant, B. C., et al., 1998, Hepatology 28:179-191; Zhang, et al., 2003, J. Med. Virol. 71:24-30) and also in human immunodeficiency virus (Lewis-Hall, F. C, 2007, Re: Important information regarding BARACLUDE (entecavir) in patients co-infected with HIV and HBV. Food and Drug Administration MedWatch Program. Food and Drug Administration, Washington, D.C.). Resistance can have broader consequences because of the extensive gene overlap in HBV, since some reverse transcriptase mutations lead to surface protein that is insensitive to antibodies generated by the HBV vaccine (Torresi, J., et al., 2002, Virology 293:305-313). During the course of treatment and especially where liver transplant is indicated, it may be necessary to evaluate the activity of HBV and thereby determine the extent of infection in the liver. One way of demonstrating the activity of the virus is to stain liver biopsies for core protein and an HBV surface antigen (HBsAg). Also important are tests for HBV cores, virus and e-antigen in serum. Currently, most HBV activity measurements and/or HBV-related tests are performed using antibody-based assays.
It has been found that heteroaryldihydropyrimidines (HAPs) enhance the rate and extent of HBV capsid protein assembly over a broad concentration range leading to aberrant particles, dominated by hexagonal arrays of capsid protein. HAPs also stabilize viral cores, thereby preventing normal dissociation and release of the viral genome (Stray, S. J., et al., 2005, Proc. Natl. Acad. Sci. USA 102:8138-8143). It is believed that such compounds disrupt HBV assembly, altering either the timing of formation of the capsid, the stability of the capsid, or the geometry of capsid formation, and interfering with viral infection accordingly. Targeting assembly of the HBV capsid protein, which has no human homolog, may therefore be a powerful, general approach for developing anti-HBV therapeutics. A hydrophobic groove, or “HAP pocket,” exists as a gap at a protein-protein interface within the HBV core (Bourne et al. 2008). When that groove, or HAP pocket, is filled, either by HAP molecules, propenamides or by core protein mutations, the effect is antiviral. Thus far, all known HBV-specific core protein assembly effectors bind within the HAP pocket. Accordingly, the availability of fluorescent-HAP stains (compounds) having high affinity for the HAP pocket of HBV would enable competitive binding assays as a means by which to identify molecules as antiviral candidates.
Fluorescent-HAP stains (compounds) for core protein would also be of interest to those studying HBV biology, e.g., in tracking HBV cores in cells and organs. The life cycle of HBV is not well defined. Numerous questions exist pertaining to localization of assembly sites, sites where cores accumulate, and core interaction with exocytotic machinery. The availability of fluorescent-HAP stains (compounds) for addition to a culture medium that would diffuse into cells and accumulate at sites where there are HBV cores would allow ready identification of localization sites for HBV cores in cultured cells. In animals (e.g., mice, woodchucks, humans), such stains could be used to identify infected regions of liver and possibly other organs where HBV cores accumulate, as anticipated for “occult” infections.
The signature of an active HBV infection is the presence in serum of HBV cores and core-containing Dane particles (i.e., HBcAg or c-antigen). These are currently detected in clinical assays using antibodies. The availability of fluorescent-HAP stains (compounds) would offer an alternative that is highly specific for HBV cores. Likewise, the presence or absence of e-antigen, i.e., the aforementioned variant of HBV core antigen, is a marker for actively replicating HBV. Infections that are c-antigen positive, e-antigen negative are associated with severe progressive chronic infections. Antibody-based assays for e-antigen have had difficulty with cross-reactivity with HBV cores (i.e., c-antigen). Adjusting the conditions of a serum sample (e.g., raising ionic strength) would be expected to render the e-antigen readily detectable by a fluorescent-HAP stain (compound).
Rapid, effective tests for HBV cores in tissue enabled by the availability of fluorescent-HAP stains (compounds) would be valuable for examining biopsies and other tissue samples for staging and grading infection liver damage, would have diagnostic value for examining patients with ambiguous clearance of acute infection, would allow evaluation of transplant candidates for the level of infection and for HBV in other targeted organs, and permit examination of samples from recent transplantees for evidence of reinfection. Thus, direct detection of HBV infection in concert with histological examination would be of value for considering treatment options for patients that have liver damage but minimal evidence of ongoing infection.
Notwithstanding the current availability of antibody-based HBV activity measurements and/or HBV-related tests, there remains a need for i) a clinical alternative to antibody-based HBV diagnostic reagents, ii) effective HBV-tracking agents, and iii) new tools for anti-HBV drug discovery, all of which needs would be supported by the availability of fluorescent-HAP stains (compounds).